Mining Sudanese Medicinal Plants for Natural Compounds against Malaria and Neglected
Tropical Diseases
INAUGURALDISSERTATION
zur
Erlangung der Würde eines Doktors der Philosophie
vorgelegt der
Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel
von
Abdelhalim Babiker Mohamed Mahmoud aus dem Sudan
Basel, 2020
Originaldokument gespeichert auf dem Dokumentenserver der Universität Basel edoc.unibas.ch
auf Antrag von
Prof. Dr. Pascal Mӓser Prof. Dr. Thomas J. Schmidt
Basel, den 23.06.2020
Prof. Dr. Martin Spiess Dekan
To my father
Who taught me that perseverance and hard work always pays off.
May your inspiring soul rest in peace.
Acknowledgment ... I Abbreviations ... III Summary ... V
1. INTRODUCTION: Neglected Tropical Diseases, Drug Discovery, and the Sudan ... 1
1.1 Neglected Tropical Diseases ... 3
1.1.1 NTDs and Sudan ... 4
1.2 Drug Discovery for NTDs: ... 6
1.2.1 Challenges and Gaps ... 6
1.2.2 Drug Development Strategies for NTDs: ... 7
1.3 Ethnomedicine in Sudan and Biodiversity ... 12
1.4 Objectives ... 14
1.5 References ... 16
2. Mining Sudanese Medicinal Plants for Antiprotozoal Agents ... 23
2.1 Abstract ... 25
2.2 Introduction ... 27
2.3 Results... 29
2.3.1 Review of medicinal plants from Sudan ... 29
2.3.2 Testing for antiparasitic activity ... 32
2.3.3 Two-way clustering of the bioactivity data ... 33
2.3.4 Testing for cytotoxicity ... 35
2.3.5 Extracts with selective anti-trypanosomal activity ... 36
2.3.6 Extracts of selective antiplasmodial activity ... 36
2.3.7 HPLC-based activity profiling ... 37
2.3.8 Dereplication of active principles ... 38
2.4 Discussion ... 42
2.5 Material and Methods ... 45
2.6 References ... 50
2.7 Supporting Information: ... 58
3. HPLC-Based Activity Profiling for Antiprotozoal Compounds in Croton gratissimus and Cuscuta hyalina ... 69
3.2 Introduction ... 72
3.3 Results and Discussion... 74
3.3.1 Extraction and HPLC-based Activity Profiling ... 74
3.3.2 Compound Isolation and Structure Elucidation ... 75
3.3.3 Activity against Leishmania donovani Axenic and Intracellular Amastigotes ... 77
3.3.4 Activity against Trypanosoma brucei rhodesiense ... 78
3.3.5 Activity against Plasmodium falciparum ... 79
3.3.6 Correlation between Chemical Structure of Isolated Flavonoids and Antiprotozoal Activity……….………79
3.4 Materials and Methods... 81
3.5 References ... 91
3.6 Supporting Information ... 95
4. Lignans, Amides, and Saponins from Haplophyllum tuberculatum and their Antiprotozoal Activity ... 113
4.1 Abstract ... 115
4.2 Introduction ... 116
4.3 Results and Discussion... 117
4.3.1 Extraction and HPLC-based Activity Profiling ... 117
4.3.2 Compound Isolation and Structure Elucidation ... 118
4.3.3 Comparison to previously reported compounds ... 121
4.3.4 Biological testing ... 122
4.3.5 Activity against Leishmania donovani axenic amastigotes ... 123
4.3.6 Activity against Plasmodium falciparum ... 123
4.3.7 Activity against Trypanosoma brucei rhodesiense ... 124
4.4 Materials and Methods... 125
4.5 References ... 134
4.6 Supporting Information: ... 139
5. Natural Products Against Madurella mycetomatis ... 151
5.1 Abstract ... 153
5.2 Introduction ... 154
5.3 Results and Discussion... 156
5.4 Materials and Methods... 162
6. In vitro testing of redox-active parasiticides identifies niclosamide as a hit for Madurella
mycetomatis and Actinomadura spp. ... 169
7. GENERAL DISCUSSION ... 183
7.1 Overview of the research outcomes ... 185
7.2 Why phenotypic screening? ... 188
7.3 Why an ethnobotanical approach? ... 189
7.4 Caveats ... 190
7.5 Extraction procedures ... 190
7.6 Bioassays and screening procedures ... 191
7.7 Dereplication ... 194
7.8 HPLC-based activity profiling ... 195
7.8.1 Considerations in the HPLC-activity profiling approach ... 196
7.9 The empirical antiparasitic drug discovery approach applying biological screening and activity-oriented separation of medicinal plants: Could it be improved? ... 197
7.10 Other options for exploring traditional medicine: lessons from history ... 198
7.11 Mycetoma Drug Discovery ... 199
7.11.1 Natural products against Eumycetoma ... 200
7.11.2 Repurposing approach ... 200
7.12 Final conclusion ... 201
7.13 References ... 203
Curriculum Vitae ... 209
I
Acknowledgments
First and foremost I offer my sincerest gratitude and heartfelt appreciation to Pascal Mäser, my supervisor, for giving me the opportunity to carry out the presented work. His unlimited support, open-minded leadership, and kindness that was reflected in both research and on a personal level at all times of my PhD gave me the courage, inspiration, and motivation to accomplish this thesis. I could not have imagined having a better advisor and mentor for my PhD study.
I would like also to thank Prof. Dr. Matthias Hamburger, for accepting me as a visiting scientist and allowed me to carry out the phytochemical investigations. His long experience, his sense of precision and detail has broadend my knowledge and my sense of analysis.
Very personal thanks go to Prof. Dr. Sami Ahmed Khalid whose constant encouragement, experienced advice and in-depth discussions guided me along the way of my research. His thorough knowledge and expertise in phytochemistry and pharmacology is a constant source of inspiration for me.
I warmly thank Dr. Marcel Kaiser for the stimulating discussions and his generous assistance and advice on the biological assays, from which I have learned and benefited a lot.
I would like to express my gratitude to Prof. Thomas J. Schmidt for joining the PhD committee and accepting to be co-referee, as well as for his time and inputs.
I am very grateful to Prof. em. Marcel Tanner and Prof. Suad Sulaiman for their mentorship and bringing this fruitful collaboration and network between Khartoum and Basel. I would also like to thank them for their continuous encouragement and support.
Many thanks to Monica Cal, Sonja Keller and Romina Rocchetti for their helpful and expert technical assistance in the bioassays performed. I’m further very thankful to Shereen Abd Algaffar for the mycetoma work that has been done in Sudan and Ombeline Danton for the
II
assistance in NMR and structure elucidations. Without their precious support it would not be possible to conduct this research.
Deep thanks extend to Christine Mensch, from the department of education and training at the SwissTPH for her continuous support and making my stay in Basel very pleasant.
My warm thanks to all current and former colleagues of the Parasite Chemotherapy Unit of the Swiss TPH and of the division of Pharmaceutical Biology. It was a great pleasure to work with you in a positive and familiar atmosphere. Sincere thanks to Natalie Wiedemar, Anna Fesser, Teresa Faleschini and Antoine Chauveau for the friendship and all the joyful moments and interesting discussions.
Very special thanks and heartfelt gratitude to my family: my mother, without your love, encouragement and whole-hearted prayers, I would not have reached this far. To my brother Mohamed, his family, my two sisters Saba and Rana, my whole extended family, and my dear friends who have given me their unequivocal support and encouragement which have paved the way to reach this stage.
Last but not least, to my wife Amal for whom my sincere expression of thanks and gratitude does not suffice. Thank you for your love, care, constant support and embracing. Throughout this journey you were my light during dark nights and my hope in desperate moments. You are my angel.
III
Abbreviations
ACT Artemisinin-based Combination Therapies COSY COrrelation SpectroscopY
DALYs Disability-Adjusted Life Years
DNDi Drugs for Neglected Diseases Initiative ECD Electronic Circular Dichroism
ELSD Evaporative Light Scattering Detector ESI ElectroSpray Ionization
GDP Gross Domestic Product
HAT Human African Trypanosomiasis
HMBC Heteronuclear Multiple Bond Correlation HPLC High Performance Liquid Chromatography HSQC Heteronuclear Single Quantum Coherence HTS High Throughput Screening
IC50 50% Inhibitory Concentration MMV Medicines for Malaria Venture
MS Mass Spectrometry
NCEs New Chemical Entities NMR Nuclear Magnetic Resonance
NOESY Nuclear Overhouser Enhancement SpectroscopY
NPs Natural Products
NTDs Neglected Tropical Diseases PDA Photodiode Array Detector
RP Reverse Phase
SAR Structure-Activity Relationship
SI Selectivity Index
UV Ultraviolet
WHO World Health Organization
iv
V
Summary
Tropical parasitic diseases such as malaria, human African trypanosomiasis, Chagas disease, mycetoma, and leishmaniasis affect more than a billion people worldwide and have devastating consequences. There is no vaccine for any of these diseases, and the current drugs are problematic given their serious adverse effects and the emergence of drug-resistant parasites.
Thus, there is an urgent need for the development of new, efficacious, safe, and cost-effective drugs.
Natural products have in many instances provided new leads to combat neglected tropical diseases. The aim of this work was to systemically evaluate Sudanese medicinal plants for their antiparasitic activity along with their cytotoxicity profile; followed by phytochemical investigation to identify bioactive compounds. A library of 235 plant extracts was prepared from over 60 plants used in Sudanese traditional medicine, and it was assessed for antiprotozoal activity against Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani, and Plasmodium falciparum.
Dereplication was performed for active extracts to enable a rapid identification of known active compounds and prioritization for follow-up isolation. Plants that displayed interesting activities, namely Croton gratissimus, Cuscuta hyalina, and Haplophyllum tuberculatum, were further pursued. HPLC-based activity profiling led to localization of activity and identification of the types of compounds in these plant extracts. Compound isolation and structure elucidation were achieved by a combination of analytical, preparative, and semipreparative chromatographic techniques such as HPLC-PDA-ELSD-MS and microprobe NMR.
HPLC-based activity profiling of Croton gratissimus allowed the identification of flavonoids, mainly quercetin derivatives, as responsible for the antileishmanial activity of the chloroform fraction of the crude ethanolic extract. Of these compounds, quercetin-3,7-dimethylether and ayanin were the most active against the protozoan parasites and with the highest selectivity indices.
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Compounds that displayed moderate to higher antitrypanosomatid activity shared structural features, such as Δ2,3 unsaturation, presence of a hydroxyl group at C-3, a carbonyl group at C-4, and a catechol moiety in ring B. Phytochemical characterization of Cuscuta hyalina lead to the isolation of a unique flavonoid, pseudosemiglabrin, for the first time from Cuscuta species.
The antileishmanial activity of Haplophyllum tuberculatum was tracked by HPLC-based activity profiling, and eight compounds were isolated from the chloroform fraction. These included the lignans tetrahydrofuroguaiacin B, nectandrin B, furoguaiaoxidin, and 3,3′-dimethoxy-4,4′- dihydroxylignan-9-ol; and four cinnamoylphenethyl amides, namely dihydro- feruloyltyramine, N-trans-feruloyltyramine, N,N′-diferuloylputrescine, and 7′-ethoxy- feruloyltyramine. The water fraction yielded steroidal saponins. All these compounds were reported for the first time from Haplophyllum species and the family Rutaceae. Nectandrin B exhibited the highest activity against L. donovani (IC50 4.5 µM) and the highest selectivity index (25.5).
Given the urgent need for better drugs and the fact that mycetoma is the most neglected of the neglected diseases, mycetoma has received special consideration. Different approaches were tackled to ultimately identify potential hits. With regard to antimycetomal natural products, several compounds were selected based on an educated-guess and were assessed accordingly.
Of the tested natural compounds, magnolol possessed the highest activity (MIC of 15 µM) and selectivity (SI of 4.9).
In parallel, a drug repurposing (repositioning) strategy was pursued to find more promising hits.
A series of nitroimidazole compounds were screened in vitro against the fungus Madurella mycetomatis. From this screening, niclosamide showed interesting activity with a minimal inhibitory concentration ˂5 µM. Furthermore, additional niclosamide analogues were tested for proof of concept. The tested compounds showed similar activity compared to niclosamide, not only against M. mycetomatis but also against the bacteria Actinomadura spp. The finding that a drug like niclosamide, which is on the WHO's list of Essential Medicines, exhibits in vitro activity against both fungal and bacterial mycetoma warrants the consideration of niclosamide or its ethanolamine salt as repurposing candidates for mycetoma.
1. INTRODUCTION:
Neglected Tropical Diseases, Drug Discovery, and the Sudan
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1.1 Neglected Tropical Diseases
Neglected tropical diseases (NTDs), as classified by the WHO, are a group of 18 chronic disabling infections caused either by viruses, bacteria, fungi, protozoa or helminths. The diseases affect more than a billion people world-wide, mainly in Africa and mostly those living in remote rural areas, urban slums, or conflict zones [1]. The burden of these diseases has a high impact in terms of human suffering as well as contributing to poverty and under-development. NTDs account for 48 million disability-adjusted life years (DALYs) and 152 000 deaths per year [2,3].
Three of these diseases are Human African Trypanosomiasis (HAT) caused by Trypanosoma rhodesiense spp., Leishmaniasis caused by Leishmania spp., and Chagas disease (American trypanosomiasis) caused by Trypanosoma cruzi [4]. The three pathogens belong to the trypanosomatidae, a large family of flagellated protozoa. Although malaria, caused by the apicomplexan parasite Plasmodium falciparum, is no longer considered an NTD since 2000, owing to increased funding level globally by various international bodies and philanthropic organizations (e.g. the Global Fund, Bill and Melinda Gates Foundation, and the Medicines for Malaria Venture (MMV)), the disease still remains a major challenge due to the heavy death toll and its negative economic impact, which translate to 1.3% annual loss in gross domestic product (GDP) in malaria endemic African countries [5]. In addition, the occurrence of the disease among the poor is disproportionate with high mortality levels among pregnant women and children [6]. In the context of the present work, malaria will be addressed among the NTDs.
General information about the above mentioned NTDs is summarized in Table 1.
Mycetoma was recently included in the WHO list of NTDs [7]. It is one of the most neglected diseases at all levels. Mycetoma is a chronic, progressively destructive morbid inflammatory disease acquired by traumatic inoculation of certain fungi (Eumycetoma) or bacteria (Actino- mycetoma) into the subcutaneous tissue [8]. The disease is geographically distributed through what is called as “the Mycetoma belt”, which includes India, Yemen, Somalia, Sudan, Senegal, Mexico, Venezuela, Colombia, and Argentina [9]. Usually the foot is the most affected part but any part of the body can be involved [10]. Late chronic stages of the disease result in destruction, deformity and loss of function and often lead to amputation.
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Table 1: Summary of Neglected Tropical Disease under the scope of the study
HAT Chagas disease Leishmaniasis Malaria Mycetoma
Causative agent
Trypanosoma brucei
rhodesiene, T. b.
gambiense
Trypanosoma cruzi
Leishmania spp.
(~21 species)
Plasmodium falciparum, P.
vivax, P.
malarie, P.
ovale, and p.
knowlesi
˃ 50 species, mainly Madurella mycetomatis (Fungal type) and
Actinomadura madurae (bacterial) Vector Glossina spp.
(tsetse fly)
Triatomine spp.(Kissing bug)
Phlebotomus
spp.(Sandflies) Anopheles spp. Unknown Geographic
distribution
Sub-saharan Africa
South and Central America
Africa, Asia, Europe, South and Central America
World-wide
Between the latitudes 15° S and 30° N
DALYs 560 000 546 000 3.32 million 82.67 million Unknown
Deaths 9 100 10 300 51 600 445 000 [11] Unknown
Treatment
Melarsoprol Eflornithine+
nifurtimox Fexnidazole
Nifurtimox Benznidazole
Liposomal amphotericin B Miltefosine Paromomycin
Artemisinin combination therapy
Fungal:
Itraconazole Bacterial:
Amikacin+ Co- trimoxazole DALYs: Disability-adjusted life years
1.1.1 NTDs and Sudan
According to WHO reports, of the 17 neglected diseases, 9 are a recognized public health problem in Sudan (Figure 1). These include: leishmaniasis, schistosomiasis, lymphatic filariasis, onchocerciasis, trachoma, guinea worm, mycetoma, soil transmitted helminths, and leprosy.
Large populations living in rural areas are infected by one or more of these diseases, with the school-age children being the most affected [12]. Sudan had made large progress in the eradication of dracunculiasis (guinea-worm disease). However, the country is still endemic for schistosomiasis and trachoma (3.6 million cases), and it has the highest incidence for cutaneous and visceral leishmaniasis in sub-Saharan countries with 15,000–20,000 new cases annually
5
[13]. The situation for mycetoma is not any better. Sudan is considered among the highest infected countries with more than 6000 cases, of which, 64% are under the age of 30. Of the mycetoma cases reported, 70% are eumycetoma, stressing the high need for effective treatment and adequate preventive and control measures to reduce the disease morbidity and mortality [14]. For malaria, Sudan is considered a high-burden and high-risk country. In 2012, more than 5000 cases were reported [15]. Malaria accounted for a higher mortality burden than disability, with an estimated total number of 44000 deaths in 2002 in Sudan [16].
Figure 1: Burden of Neglected Tropical Diseases with emphasis on Sudan.
(Source: https://www.cdc.gov/globalhealth/ntd/diseases/ntd-worldmap-static.html)
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1.2 Drug Discovery for NTDs:
1.2.1 Challenges and Gaps
Most of the currently available drugs for NTDs have drawbacks in terms of toxicity, limited availability of oral therapeutic dosage forms, development of resistance, or non-affordability, coupled with unavailability of vaccines for any of them. However, drug discovery and development of new and better medicines for NTDs is costly and of low-return. As already reported, of the 1602 new chemical entities (NCEs) that has been approved between 1981 and 2019, unfortunately, only 20 medicines out of the total were for the treatment of neglected diseases [17]. The creation of public-private partnerships like DNDi (Drugs for Neglected Diseases initiative) has helped to overcome this bottleneck. A sustainable solution for new drug development became feasible with the backing support of international pharmaceutical policy and collaboration [18].
Nevertheless, there are still major gaps and so far, with the exceptions of tafenoquine for malaria and fexinidazole for HAT, these initiatives have brought to market mainly new formulations and combinations of already existing drugs.
While the incidence of human African trypanosomiasis is at a historic low and a new oral drug, fexinidazole [19], has recently received positive opinion by the European Medicines Agency, the prospects are gloomier and less good for Chagas' disease and leishmaniasis. In Eastern Africa, a high-burden region of visceral leishmaniasis [20], sodium stibogluconate is still the mainstay of leishmaniasis chemotherapy [21], a pentavalent antimonial that can cause hepatotoxicity and cardiotoxicity [22]. There is one efficient and safe antileishmanial drug, AmBisome, a liposomal formulation of amphotericin B that was developed as a fungicide and repurposed for leishmaniasis [23]. However, AmBisome is expensive and requires a cold chain to delivery.
Hence, many reports have outlined research priorities for kinetoplastids parasites regarding the need of new cost-effective therapies as a key element of the fight against protozoal neglected tropical diseases [24,25].
The treatment of malaria still relies globally on artemisinin-based combination therapies (ACT).
Alarmingly, artemisinin-resistant isolates of P. falciparum have been described from south-east Asia [26–28] and most recently also from Africa [29]. Thanks to an increased funding level
7
globally by various international bodies and philanthropic organizations such as Medicine for Malaria Venture (MMV), there is a series of new molecules in the drug discovery pipeline.
However, none of these has reached registration yet.
The bacterial type of the mycetoma, actinomycetoma, is readily cured by antibiotics combination therapy [30]. In contrast, management of the fungal type (Eumycetoma) is much more difficult. Treatment usually involves surgical excision combined with long term use of azole antifungals, which have limited efficacy, toxic adverse effects, are expensive, and have with high percentage of treatment failures [31]. DNDi together with the Mycetoma Research Center in Khartoum are currently running a phase II/III clinical trial to assess the efficacy and safety of fosravuconazole in comparison to the currently used itraconazole. Nevertheless, there is a dire need to find new therapeutic agents for eumycetoma that are efficient, affordable, safe, and decrease treatment period and surgical interventions [32].
1.2.2 Drug Development Strategies for NTDs:
Strategies for hit discovery usually involve two opposing, yet complementary, screening pathways; target-based (typically a protein or an enzyme), or phenotypic screening (whole organism, cell-based). Despite the paradigm shift in the pharmaceutical industry to move from whole-cell to target-based screening, in the case of antiparasitic drugs, the phenotypic approach has proven to be more successful. This success could be explained by the facts that (i) it does not require prior knowledge of the molecular target, which is the case of most neglected parasites where there are very few validated molecular targets; (ii) it enables high throughput screening of chemical libraries and identification of chemical entities without a known target or mechanism of action; (iii) target deconvolution is possible with the aid of genomic tools; (iv) successful drug candidates are likely to involve interaction with a number of different target enzymes ("unspecific" mode of action), which is the case of most of the currently used antiparasitics [33–35].
Alternative approaches include structure-based drug discovery, re-purposing of drugs from other disease areas, and in silico methods. Each strategy has its own advantages and disadvantages.
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Of the many avenues and possibilities of drug discovery for neglected tropical diseases [36,37], two strategies will be highlighted and discussed in the scope of this work to fill the drug pipeline against these devastating and global diseases. These are; i) repurposing, and ii) natural products.
1.2.2.1 Repurposing
Since drug development is lengthy and expensive, the drug repurposing strategy (i.e. finding new uses for existing drugs) offers an attractive shortcut between the bench and the clinic, particularly where the resources for R&D are limited [38]. The concept of repurposing is actively pursued for NTDs, and many drugs that are currently used for the treatment of neglected tropical diseases have been ‘repositioned’ (Table 2). Concurrently, most of the repurposed compounds have arisen from phenotypic screening campaigns rather than target-based strategies [39], Owing to the limited number of fully validated targets in NTDs, as discussed earlier. Repurposing screening campaigns for NTDs have revealed potential molecules of different drug classes, like tricyclic antidepressants for antipalsmodial activity, tadalafil and the antispasmodic mebeverine for Chagas’ disease, along with other molecules that fit established criteria of Target Product Profiles (TPP) for NTDs [40]. Interestingly, not only drugs used in other diseases are repurposed for NTDs, but also the other way around: suramin, developed for Nagana and sleeping sickness, was repurposed for the treatment of cancers and autism [41], and an exciting case is the antimalarial chloroquine in clinical trials for the treatment of the recent virus pandemic COVID-19 [42]. In the context of this work, nitroimidazoles were tested for their in vitro activity against Madurella mycetomatis, the major causative agent of Eumycetoma.
Table 2: Drugs repurposed for NTDs
Drug Original use Repurpose Reference
Eflornithine Anticancer HAT [43]
Ivermectin Onchocerca in horses River blindness [44]
Fosmidomycin Antibiotic Malaria [45]
Doxycycline Antibiotic Filariasis and Malaria [46]
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Amphotericin B Antifungal Visceral leishmaniasis [47]
Miltefosine Anticancer Visceral leishmaniasis [47]
Paromomycin Antibiotic Visceral leishmaniasis [47]
Pentamidine Equine trypanosomiasis HAT- Stage 1 [43]
Albendazole Anthelmintic for livestock Lymphatic filariasis [48]
Nifurtimox Chagas disease HAT [49]
1.2.2.2 Natural Products
Natural products (NPs) remain a successful source of inspiration for the discovery of new drugs.
A recent comprehensive review by Newman and Cragg, covering approved drugs during the period 1981-2019, revealed that one third of the small molecules launched over the last four decades were derived directly or indirectly from natural resources. Moreover, of the 20 approved antiparasitic drugs, 9 were of natural origin [17], some of them are summarized in Table 3. Many chemo-informatic studies showed that natural products cover a much wider and larger chemical space than combinatorial and synthetic compounds, due to their diversity in terms of chiral centers and richness in functional groups, which render them viable for a wider ligand affinity and better specificity to biological targets [50,51].
Table 3: Examples of antiparasitic drugs of natural or derived from natural origin
Drug Natural origin Source Classification Reference
Artemisinin Artemisia annua Plant N [52]
Ivermectin Streptomyces avermitilis Bacteria N [53]
Quinine Cinchona succirubra Plant N [54]
Moxidectin Milbemycin derivative from
Streptomyces cyanogriseus spp. Bacteria ND [55]
Eflornithine Difluoromethyl derivative of
ornithine Amino acid ND [56]
N: natural product; ND: natural product derivative
Many secondary metabolites with a wide variety of scaffolds, namely alkaloids, terpenes, and phenolic compounds (e.g. lignans, tannins, coumarins, flavonoids) have shown potent inhibition
10
of parasites responsible for NTD [37,57–59]. However, the potent activities displayed by some of these NPs are hampered by their toxicity and pharmacokinetic profiles that prevent their use in the clinic. Nonetheless, these hits can be modified by medicinal chemistry and drug delivery approaches to enhance the pharmacokinetic and safety characteristics.
1.2.2.3 Challenges and opportunities of Natural Products Drug Discovery
Despite the success of NPs, the interest of several major pharmaceutical companies has waived, and they cut down the use of natural products in their drug discovery programs. A major concern is that NPs are incompatible with high throughput screening (HTS), laborious to handle, highly complex, have non-specific activities, and issues with accessibility, logistics, and patentability [60]. A more rational and economic search for new lead structures from nature must therefore be a priority in order to overcome these problems. Key factors to achieve this competitiveness include employment of technological advances like robotics, bioassay miniaturization, and developments in spectroscopy in the NPs-based lead discovery processes such as speed of dereplication, bioassay-guided isolation, and structure elucidation [61].
Innovative omics-based approaches integrated with molecular networking enabled the prioritization and targeted isolation of novel natural products, and provided means to bridge the gap between ethnopharmacological drug discovery and industrial biotechnology for monitoring fermentation or other production processes [61–63].
1.2.2.4 Strategies for identification and Isolation of Bioactive Natural Products
A variety of approaches are being used for identification of bioactive secondary metabolites from plant, fungal, microbial, or marine sources, such as: i) traditional medicine and ethno- pharmacological knowledge (antimalarials, quinine and artemisinin), ii) taxonomical - chemotaxonomical (anticancer, taxol), iii) ecology-based (marine natural products, insecticides, antifeedants), and iv) pharmacophore-based or virtual screening (computer-based) [64].
The process of “bioassay-guided fractionation” starts once an extract has shown favorable activity in a screening. Then, it is necessary to isolate the compound(s) responsible for the pharmacological properties. Since extracts are complex matrices, there is the challenge of
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localizing activity in the extract by overlaying biological data and chemo-analytical information in order to identify the active principles at an early stage [65]. One of these approaches is dereplication. Dereplication was initially defined as “the process of quickly identifying known chemotypes’’ [66]. The definition has developed and extended over the years to include many strategies with the ultimate goal of accelerating the discovery of bioactive substances by improving the characterization methods of natural resources. Dereplication allows elimination and prioritization of extracts by comparing the chemical and biological characteristics of unknown compounds to that of previously identified compounds in databases. Hence, comprehensive databases are crucial for high performance dereplication workflows [67,68].
Another approach is HPLC-based activity profiling, which has been successfully used for tracking bioactive compounds in crude mixtures [69].The principle of this approach consists in the analytical scale HPLC separation of bioactive extracts. UV and MS data are recorded online in parallel to collection of fractions into microplates or deep-well plates, via a T-split of the column effluent. The fractions are dried, re-dissolved in a small amount of a suitable solvent (usually DMSO) and assayed for bioactivity. The chromatogram and the activity profile are then matched to identify active peaks. On-line spectroscopic information in combination with database searches can be used to dereplicate known compounds and facilitates prioritization of samples for follow-up activities [70,71]. (Figure 2)
12 Figure 2: HPLC-based activity profiling approach
1.3 Ethnomedicine in Sudan and Biodiversity
Sudan is the third largest country in the African continent (after Algeria and Democratic Republic of the Congo). Located in east central Africa with a total surface area of 1.8 million km2, Sudan encompasses different terrains and climatic zones, ranging from desert and semi- arid in the north to tropical savanna in the south. The country consists of a vast flat landscape bordered by mountains on the north east (the Red Sea Hills) and the west (the Marrah Mountains). The northern part features the Nubian Desert (part of the Sahara desert) and the east part reaches out to the red sea (Figure 3). This matchless geographical topography of Sudan, with its variable climates, makes it a unique place with different ecosystems and a richness of plant biodiversity.
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Figure 3: Topographical map of the Sudan, desert in the North and main mountain ranges: Red sea hills in northeast and Marrah mountains in the west.
(Source: https://www.nationsonline.org/oneworld/map/sudan-topographic-map.htm)
The flora of Sudan consists of 3137 species of flowering plants belonging to 170 families and 1280 genera [72]. It is estimated that 15% of these plants are endemic to Sudan. The
intersection of diverse African, Arabic and Islamic cultures influenced the uniqueness of folkloric and herbal medicine [72]. Sudanese traditional medicine is an indigenous form of holistic health care system involving both mind and body (i.e. psychosomatic medicine). It is a unique combination of natural, cultural and religious background that prevalent in the
community. However, there is limited published data available on the biological activities of the Sudanese medicinal plants.
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1.4 Objectives
Sudan's biodiversity of medicinal plants coupled with deeply rooted ethno-botanical heritage remains a promising untapped reservoir for the discovery of diverse chemical entities. The main goals of this Ph.D. thesis are:
1. Better characterization of Sudanese medicinal plants and a rationale for their use.
2. Finding promising antiparasitic hit compound(s) that have the potential to generate novel leads.
3. Exploring different approaches of drug discovery for the neglected disease Mycetoma.
Chapter 2 starts with a comprehensive overview on medicinal plants that are being used traditionally in Sudan for tropical illnesses, with a focus on protozoal diseases and plants that were pharmacologically investigated. On the basis of this survey, a library consisting of 235 extracts andfractions thereof, representing 62 plants reputed as antiparasitics that belong to 35 different plant families, was assembled, prepared, and screened phenotypically for in vitro activity against the following panel of protozoal parasites: T. b. rhodesiense bloodstream form, T. cruzi intracellular amastigote form grown in rat L6 cells, L. donovani axenic amastigote form grown at low pH, and P. falciparum proliferative erythrocytic stages grown in human erythrocytes. For selected active extracts, HPLC-based activity profiling in combination with on- line spectroscopy enabled a rapid identification of some of the bioactive compounds by dereplication.
Chapter 3 and Chapter 4 continue with phytochemical characterization using the HPLC- activity profiling approach for plant extracts of promising antiprotozoal activity, followed by the isolation of their bioactive compounds by different preparartive and semipreaparative chromatographic techniques, and finally elucidation of the chemical structures. The activity of the isolated compounds was determined in vitro (whole-cell assays), alongside with cytotoxicity testing in mammalian cells. The main purpose of the cytotoxicity test was to calculate the
15
selectivity index (SI), which allows discriminating attractive compounds from poorly selective ones.
Chapter 5 investigates the In vitro antimycetomal activity, together with cytotoxicity profiling of compounds isolated from the chloroform and the water fractions of the ethanolic extract of Haplophyllum tuberculatum roots (Forsskal) A. Juss. (Rutaceae). In addition, various natural compounds of different calsses of plant secondary metabolites obtained from different plant species, and that have been previously reported for their antifungal and anti-infective activities, were also screened for their activity and cytotoxicity against Maduralla mycetomatis.
Chapter 6, the repurposing approach, was pursued for finding potential drug candidates that are active against fungal mycetoma. Selection and testing of nitroimidazoles and other redox- active molecules was performed based on their potential antifungal mechanism of action. The hypothesis that redox-active molecules will also be active against Madurella mycetomatis, the principal causative agent of eumycetoma, was not confirmed. Nevertheless, niclosamide was identified as a potential drug candidate.
16
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2. Mining Sudanese Medicinal Plants for Antiprotozoal Agents
Abdelhalim Babiker Mahmoud1,2,3*, Pascal Mäser1,2* , Marcel Kaiser1,2, Matthias Hamburger2 and Sami Khalid3,4
1Swiss Tropical and Public Health Institute, Basel, Switzerland.
2University of Basel, Basel, Switzerland.
3Faculty of Pharmacy, University of Khartoum, Khartoum, Sudan.
4Faculty of Pharmacy, University of Science and Technology, Omdurman, Sudan.
Published in
Frontiers in Pharmacology-Ethnopharmacology 2020;11:865.
doi:10.3389/fphar.2020.00865
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I have performed all experiments and analysis and wrote the manuscript
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2.1 Abstract
Neglected tropical diseases are major health hazards in developing countries. Annually, up to thirty million people are affected by either Chagas disease, African trypansomiasis or leishmaniasis, and more than 200 million by malaria. Most of the currently available drugs have drawbacks in terms of toxicity, limited oral availability, development of resistance, or non-affordability. Tropical plants of the arid zones are a treasure chest for the discovery of bioactive secondary metabolites. This study aims to compile Sudanese medicinal plants, validate their antiprotozoal activities, and identify active molecules.
We have performed a survey of medicinal plants of Sudan and selected 62 that are being used in Sudanese traditional medicine. From these, we collected materials such as leaves, stem, bark, or fruit. The plant materials were extracted in 70% ethanol and further fractionated by liquid-liquid partitioning using solvents of increasing polarity.
This resulted in a library of 235 fractions. The library was tested in vitro against Plasmodium falciparum (erythrocytic stages), Trypanosoma brucei rhodesiense (bloodstream forms), Trypanosoma cruzi (intracellular amastigotes), and Leishmania donovani (axenic amastigotes). Active fractions were also tested for cytotoxicity. Of the 235 fractions, 125 showed growth inhibitory activity >80% at 10 μg/mL, and >50% at 2 μg/mL against at least one of the protozoan parasites. Plasmodium falciparum was the most sensitive of the parasites, followed by T. b. rhodesiense and L. donovani. Only few hits were identified for T. cruzi, and these were not selective. Contrary to expectation based on phylogeny, but in agreement with previous results, a large number of extracts displayed mutual activity against T. brucei and P. falciparum. HPLC-based activity profiling for selected active extracts was performed to identify the bioactive principles.
Active compounds identified by dereplication were guieranone A from Guiera senegalensis J.F.Gmel.; pseudosemiglabrin from Tephrosia apollinea (Delile) DC; ellagic acid and quercetin from Anogeissus leiocarpa (DC.) Guill. & Perr.; and catechin, ethyl gallate, and epicatechin gallate from Acacia nilotica (L.) Delile. Also the extracts of Croton gratissimus var. gratissimus and Cuscuta hyalina Roth ex Schult. exhibited promising antitrypanosomatid activity. This assessment provides a comprehensive
26
overview of Sudanese medicinal plants and supports the notion that they are a potential source of bioactive molecules against protozoan parasites.
Keywords: HPLC-activity profiling, Drug discovery, Sudan, Medicinal plant, Trypanosoma, Leishmania, Plasmodium.
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2.2 Introduction
Infections by protozoan parasites remain to be among the most devastating causes of mortality in the tropics. The trypanosomatids are a large family of flagellated protozoa, some of which cause neglected tropical diseases of high public health relevance and socio-economic impact [1,2]. These are Trypanosoma cruzi (Chagas' disease), T. brucei gambiense and T. b. rhodesiense (human African trypanosomiasis or sleeping sickness), and Leishmania spp. (different kinds of leishmaniasis) [3]. The apicomplexan parasite Plasmodium falciparum is the causative agent of malaria tropica, the most dangerous form of malaria, which – despite the successes by various international bodies and philanthropic organizations – still claims an annual death toll of 435,000 (World Malaria Report, WHO 2018). These diseases disproportionally affect the poor and vulnerable populations [4], calling for action to improve global well-being. A key element of the fight against protozoan neglected tropical diseases and malaria is the discovery of novel chemotherapeutic agents.
While the incidence of human African trypanosomiasis is at a historic low and a new drug, fexinidazole [5], has recently received positive opinion by the European Medicines Agency, the prospects are slightly gloomy for other protozoal diseases. Chagas' disease has reached global dimensions [6], and leishmaniasis as well [7]. Sudan has the highest incidence of leishmaniasis in sub-Saharan countries, with 15,000–20,000 new cases per annum [8]. The successful treatment of malaria is threatened by artemisinin-resistant mutants of P. falciparum, first reported from Southeast Asia [9–11] and, more recently, also from Africa [12].
Plants are still considered as important sources for the discovery of novel bioactive molecules. Plants secondary metabolism represents a huge and unique reservoir of chemical diversity, which may serve as a source of new drugs, either directly or after optimization by medicinal chemistry. Independent chemoinformatic analyses have
28
consistently shown that natural products often exhibit unique features, a high degree of structural diversity, and drug- or lead-like structural properties [13–15].
A retrospective analysis showed that approx. 50% of drugs approved within the last 30 years are derived, directly or indirectly, from natural products, whereby plant derived compounds played an important role [16].
Sudan's biodiversity coupled with a deeply rooted ethno-botanical heritage is an untapped reservoir for the discovery of new bioactive natural products. Here we performed a survey of plants from Sudan that are used in traditional medicine, with a focus on malaria and neglected tropical diseases caused by protozoa. On the basis of this survey a library of plant extracts was assembled and screened against trypanosomatid parasites and P. falciparum. Active compounds in the most promising extracts were tracked with the aid of an activity-driven approach.
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2.3 Results
2.3.1 Review of medicinal plants from Sudan
Ethnopharmacological literature review based on scholarly databases (Pubmed, Medline, SciFinder) and other supporting documents revealed that 34 of the 62 plants had been recorded for use against leishmaniasis, trypanosomiasis or malaria, including the symptoms related to any of these diseases (Table 1). Several of the plants had also been investigated pharmacologically and had exhibited anti-infective activity (Table 2).
Table 1: Plants investigated in the present study that have a reported use as anti-infective in traditional medicine.
Plant species Family Vernacular
name
Plant
part Traditional medicinal use Abutilon pannosum var. figarianum (Webb)
Verdc. Malvaceae Humbuk,
Gargadan Leaves Malaria, hepatoprotective, antibacterial [17]
Acacia nilotica (L.) Delile Fabaceae Sunt Leaves
Malaria [18], respiratory infections, diarrhoea,
haemorrhage [19]
Ambrosia maritima L. Asteraceae Damsissa Leaves
Malaria, kidney stones, renal colic, hypertension
[20]
Anethum graveolens L. Apiaceae Shabat, Dill Fruit, seeds, oil
Colic, carminative, flatulence and dyspepsia, joint swelling, sedative for
babies, lactogenic [21]
Annona muricata L. Annonaceae Leaves Antitumor, antiparasitic [22]
Anogeissus Leiocarpa (DC.) Guill. & Perr. Combretaceae Sahab Bark Cough, dysentry, giardiasis [23]
Argemone mexicana L. Papaveraceae Leaves Malaria, early-stage
trypansomiasis [24]
Aristolochia bracteolata Lam. Aristolochiaceae Irg el Agrrab,
Um Galagil Root Malaria, scorpion stings [25]
Azadirachta indica A.Juss. Meliaceae Neem Oil Malaria, antihelminthic [26]
Boswellia papyrifera (Caill. ex Delile) Hochst Burseraceae Luban Gum Cough, respiratory infections [27]
Cardiospermum halicacabum L. Sapindaceae Leaves Malaria, antiparasitic [28]
Combretum glutinosum Perr. ex DC. Combretaceae Habeil Seeds Fever, rheumatism [29]
Combretum hartmannianum Schweinf. Combretaceae Wood
Jaundice, diabetes, rheuma, wound healing, anthelminthic [30]
